Successful pathogens use varied strategies to traverse host epithelial barriers and evade immune responses. Host cell surface glycosaminoglycan (GAG)--highly-sulfated saccharide anchored in the membrane by a protein core as a heparan sulfate proteoglycan (HSPG)--is a structure commonly targeted by microbial adhesins. The broad goals of this project are to better understand the mechanisms by which GAGs/HSPGs influence host responses to infection and to modulate GAG-binding interactions to enhance host defenses against pathogens. We will study GAG-binding interactions using a Drosophila model of streptococcal infection to take advantage of well- characterized bacterial and host mutants, minimal host genome redundancy, and feasible approaches for generating additional mutants through in vivo RNAi targeting of conserved components of the GAG synthesis pathway. Group B Streptococcus (GBS) commonly colonizes human mucosal surfaces and also causes devastating invasive infections, particularly in neonates, peripartum women, and nonpregnant adults with underlying conditions. Alpha C protein (ACP) is an important GBS virulence determinant that binds to host cell GAG and mediates bacterial entry into host cells. Our preliminary data in a Drosophila model demonstrate that the interactions between host GAG and ACP determine infection outcome. Soluble GAG from human cells protected hosts from lethal infection. Other approaches to disrupting this interaction--either by decreasing the bacterial protein's GAG-binding affinity, or by decreasing sulfated host GAG levels--resulted in longer host survival and lower bacterial colony counts after infection. We also found 1. longer survival among GAG/HSPG-mutated hosts than among wild-type hosts after infection with GAG-binding-deficient mutant bacteria, 2. decreased CNS penetration of GBS in GAG-deficient mice. We now hypothesize that 1. microbial binding to sulfated host cell GAGs allows microbes to evade or suppress host defenses, and 2. interruption of sulfated GAG-binding interactions may enhance host defenses against GAG-binding pathogens. We will test our hypotheses as follows: In AIM 1 we will determine the effects of sulfated host GAG on host-pathogen interactions in vitro and in vivo by assessing immune functions and bacterial dissemination in hosts/cells with mutations altering sulfated GAG structures. In AIM 2 we will identify GAG structure(s) that bind to ACP and determine their efficacy as preventive and therapeutic agents for GBS infection. Through these studies we will improve understanding of host GAG in infection, allowing us to design better means of predicting, preventing, and treating infections. PUBLIC HEALTH RELEVANCE: Many pathogens attach to host cell surface glycosaminoglycans (GAGs). GAG-binding to the alpha C protein of group B Streptococcus, a cause of devastating neonatal infections, promotes entry of bacteria into host cells and host death. This project focuses on understanding the mechanisms by which GAGs influence infection outcome and enhancing host defenses against pathogens by interfering with GAG-binding interactions.